RRC ID 36037
Author Bhat HB, Kishimoto T, Abe M, Makino A, Inaba T, Murate M, Dohmae N, Kurahashi A, Nishibori K, Fujimori F, Greimel P, Ishitsuka R, Kobayashi T.
Title Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains.
Journal J. Lipid Res.
Abstract A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.
Volume 54(10)
Pages 2933-43
Published 2013-10
DOI 10.1194/jlr.D041731
PII jlr.D041731
PMID 23918047
PMC PMC3770106
MeSH Amino Acid Sequence Cholesterol / chemistry Cholesterol / metabolism Conserved Sequence Fungal Proteins / chemistry* Fungal Proteins / metabolism Green Fluorescent Proteins / chemistry Green Fluorescent Proteins / metabolism HeLa Cells Hemolysin Proteins / chemistry* Hemolysin Proteins / metabolism Humans Membrane Microdomains / chemistry Membrane Microdomains / metabolism* Molecular Sequence Data Pleurotus / chemistry* Protein Binding Recombinant Fusion Proteins / chemistry Recombinant Fusion Proteins / metabolism Sequence Analysis, Protein Sphingomyelins / chemistry Sphingomyelins / metabolism Staining and Labeling
IF 4.505
Times Cited 15
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material pET28/PlyA2-EGFP-His6 (RDB13962)